Fluorescent microscopy is an important aspect in many fields, especially but not limited to applications in the life sciences. Many applications involving the detection, measurement and analysis of nucleic acids, proteins, antibodies, single or multiple cells, tissue samples, and other biological materials utilize one or more fluorescent labels for detection of desired analytes. These applications include, for example, the use of nucleic acid microarrays for analysis of copy number variation, drug metabolism analysis, genome-wide or targeted genotyping, molecular cytogenetics, resequencing, gene profiling, gene expression, gene regulation, miRNA, whole-transcript expression and profiling, and other applications. Additional applications include the analysis of proteins such as transcription factors or cytokines, nucleic acids at various levels and from various sources, including in situ with respect to single cells or tissue samples, and from cell extracts, tissue lysates, conditioned media, patient sera and plasma. Further applications involve the use of fluorescent labels as a component within various nucleic acid sequencing techniques, such as pyrosequencing, sequencing by ligation, unchained sequencing by ligation of DNA nanoballs, or sequencing by synthesis with reversible dye-terminators.
Methods, systems and apparatuses for the imaging of fluorescently labeled samples through the illumination and excitation of one or more labels and the acquisition of one or more images of the corresponding fluorescent emissions are well known in the art. For example, U.S. Pat. Nos. 5,578,832; 5,834,758; 6,025,601; and U.S. Pat. No. 6,252,236 to Trulson et al. disclose methods, systems and apparatuses for generating electromagnetic radiation of a particular wavelength, optics for focusing and directing the radiation, optics for collecting responsive radiation from fluorescently labeled samples, and assembling an image from the detected responsive radiation. Trulson et al. additionally disclose techniques for auto-focusing to maintain the sample within the focal plane of the excitation light throughout the scanning process. U.S. Pat. Nos. 5,631,734; 6,141,096; and U.S. Pat. No. 6,741,344 to Stern et al. disclose related methods, systems and apparatuses for the detection of fluorescently labeled materials within a flow cell. Techniques utilizing galvanometer mirrors as an aspect of the radiation component are discussed within U.S. Pat. Nos. 5,981,956; 6,207,960; and U.S. Pat. No. 6,597,000 to Stern. Further techniques for increasing the field of view during scanning while maintaining a high scan speed and resolution are discussed within U.S. Pat. Nos. 6,185,030; 6,201,639; 6,335,824; and U.S. Pat. No. 7,312,919 to Overbeck.
While the quality and accuracy of the resulting images of scanned fluorescent materials is dependent upon many factors, proper adjustment of instruments for calibration and alignment is important to maintaining consistent and accurate performance. For example, U.S. Pat. Nos. 7,689,022; 7,871,812; and U.S. Pat. No. 7,983,467 to Weiner et al. disclose auto-focusing techniques to determine and maintain the best plane of focus for scanning. Some of these techniques are based upon the use of calibration features, such as a chrome border, with the calibration and focusing being performed using, for example, detection of the reflected light from the calibration features. Additional techniques utilizing positional reference features to adjust and update a scanned image with respect to expected and actual positions within the image are disclosed in U.S. Pat. No. 7,406,391 to Miles. However, the use of metal features is generally associated with calibration of the scanning instrument with respect to alignment (e.g., exact positioning of the fluorescent target with respect to expected or optimal positions for illumination and/or detection) and with respect to related aspects such as the calibration of the relevant optical channel to be utilized for the alignment and positioning determinations.
Fluorescence microscopy has been and continues to be a field of significant research. Underlying principles of the excitation and emission of fluorophores, the operation of fluorescence microscopes, filtering options, potential light sources, and techniques to optimize fluorescence detection can be found in a variety of sources. (See, e.g., Lichtman and Conchello, “Fluorescence microscopy,” Nature Methods, 2: 910-919 (2005); Haustein and Schwille, “Trends in fluorescence imaging and related techniques to unravel biological information,” HFSP Journal, 1(3): 169-180 (2007); Wolf, “Fundamentals of Fluorescence and Fluorescence Microscopy, Methods in Cell Biology, 81: 63-91 (2007); Coling and Kachar, “Principles and Application of Fluorescence Microscopy,” Current Protocols in Molecular Biology, 14: 14.10 (2001); Waters and Swedlow, “Interpreting Fluorescence Microscopy Images and Measurements, Evaluating Techniques in Biomedical Research, Cell Press, pages 37-42 (2007)). The associated development of new excitation sources, detectors, imaging techniques, analysis techniques and fluorescent labeling chemistries is also widely described. (See, e.g., Michalet et al., “The Power and Prospects of Fluorescence Microscopies and Spectroscopies,” Annual Review of Biophysics and Biomolecular Structure, 32: 161-182 (2003); Wouters, “The physics and biology of fluorescence microscopy in the life sciences,” Contemporary Physics, 47(5): 239-255 (2007)). Optimization of the overall imaging system related to fluorescence microscopy, such as utilization of light traps, improved fluorescent labels and image filtration routines, continue to be explored. (See, e.g., Petty, “Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology,” Microscopy Research and Technique, 70(8): 687-709 (2007); Waters, “Accuracy and precision in quantitative fluorescence microscopy, The Journal of Cell Biology, 185(7): 1135-1148 (2009)). Many aspects of fluorescence microscopy, however, remain difficult or time consuming to optimize for particular configurations of instruments, fluorophores, imaging techniques, software and the other related aspects of fluorescence microscopy. (See, e.g., Brown, “Fluorescence microscopy—avoiding the pitfalls,” Journal of Cell Science, 120: 1703-1705 (2007); North, “Seeing is believing? A beginners' guide to practical pitfalls in image acquisition,” The Journal of Cell Biology, 172(1): 9-18 (2006)). All of the above references are incorporated herein in their entirety for all purposes.
Many fluorescent detection instruments utilize a plurality of optical channels, with one or more channels configured for the detection of fluorescent emissions from one or more types of fluorophores. Previous approaches regarding fluorescent channel calibration include U.S. Pat. Nos. 6,472,671 and 6,984,828 to Montagu, which discuss the use of specialized calibration tools, similar dimensionally to the actual objects to be utilized with the instrument, and which possess a fluorophore layer under a non-fluorescent layer which is etched to form a desired pattern. Subsequent excitation and detection of the resultant fluorescent signal through the non-fluorescent pattern allows calibration of the one or more fluorescent channels of the relevant instrument.
Accurate and precise evaluation, calibration, and testing of fluorescent channels remain difficult in many respects. For example, creating and utilizing effective, cost-efficient fluorescent calibration targets that exhibit emissions comparable in wavelength spectra and strength to the fluorescently labeled biological materials to be subsequently analyzed continues to be a need. Additionally, the creation of fluorescent calibration targets that further possess characteristics that will be effective in optimizing performance of the instrument and overall scanning system with respect to the precise requirements which will actually be utilized remains unfulfilled. Furthermore, techniques are also needed to test and calibrate fluorescent channels while also testing and calibrating any associated reflective channels of the instrument in a compact manner. This need is further complicated by the requirement to desirably maintain effectiveness for the testing of both types of channels, and is further complicated for systems designed to evaluate and calibrate two or more different fluorescent channels, as may be present in multi-color detection instruments.